Humanized Antitumor Binding Sites - Framework Engineering

Huston, James

Abstract

Antibody variable (v) regions each comprise 3 complementary determining regions (CDRs) that form the antigen binding site and 4 framework regions (FRs) that form the scaffold supporting the binding site. These segments are interspersed sequentially in each V region to form FR1-CDR-1 FR2-CDR-2-FR3-CDR3-FR4, with the whole binding site requiring VH and VL. This proposal explores the modification of the FR structure for the improvement of immunochemical and solution properties of minimal antibody binding sites, consisting of single-chain Fv (sFv, comprising VH-linker-VL) or single domain V regions. In the folded conformation of each V domain, FRs form a barrel of beta pleated sheet and the CDRs form loops that connect the inner FRs to form the antigen binding site. Fr sequences from human antibodies can be fused to CDRs (a process termed humanization (which eliminates much of the antigenicity contributed by murine antigenic sites. This proposal seeks to develop advanced FR designs to be used in making hybrid sFv or variable domains, having improved stability/solubility and reduced sFv immunogenicity. The investigators will produce a humanized 26-10 antidigoxin sFv and a murine-murine hybrid sFv with an antilysozyme binding site grafted onto the 26-10 framework. In addition, the investigators will explore minimal binding sites comprising only the 26-10 VH domain, as a monomer or single-chain homodimer. Finally, they will analyze an engineered human framework which retains only simple -gly-gly-ser-gly-gly-loops to connect FRs. This nonfunctional """"""""framework only"""""""" molecule could be a cornerstone for rapid development of very well behaved biosynthetic antibody binding sites. It would allow any FR superstructure to be directly analyzed and optimized without the perturbation of a particular binding site; specific CDRs would be reinserted for applications to tumor imaging, therapy, etc.